Prepared by: Sandip Patel Group No: 2Hemin ShahMunir ShahHiren PatelParesh PatelRajeshkumar Patel

Introduction Sources and type of contamination How contamination can be

controlled? Emerging technologies or

procedures to improve yield Example of batch failure Conclusion Reference

Complex biopharmaceutical process Factor affecting cost

(source) ([9] Seymour, May 22, 2006) ([8] Scott M. Wheelwright)

Huge investment wants profit

(source) ([4] Langer, Aug 1 2008 (Vol. 28, No. 14))

Batch failureCauses

Statistics

(source)([4] Langer, Aug 1 2008 (Vol. 28, No. 14))

Consequences Most Obvious Less Obvious

Major cell contaminants Biological Chemical

Contaminant General Indications Microscopic Appearance Common Source

Bacteria pH change

Turbidity/Cloudiness

Precipitation

Fine Granules Cell culturist

Water bath

Yeast Cloudiness

pH change

Oval organisms

Budding/chains

Airborne

Fungus Spores

Furry Growths

pH change

Thin spores Airborne

Mycoplasmas Often covert

Poor cell adherence

Reduced growth

- Cell culturist

Other cell lines

Virus Sometimes cytopathic - Serum

Cell lines

• Protozoa• Invertebrates

Source: Reference [3]

Source: Reference [18]

# Culture Tested # Positive

Netherlands 1,949 488 (25%)

Czechoslovakia 327 121 (37%)

Argentina 65%

Japan 80+%

Israel 32%

# Cultures Tested # Positive

FDA (1970’s to 1990’s) (11)

20,000 Cultures Tested

Over 3000 (15%)

Bionique Testing Laboratory (several years prior to 1993) (41)

11,000 Culture Tested 1218 (11.1%)

Microbiological Associates (1985 to 1993) (13)

2,863 Culture Tested

370 (12.9%)

American Type Culture Collection (ATCC) (1989 to 1994) (42)

5,362 Culture Tested 752 (14%)

Source: Reference [7]

Contact with non sterile supplies, media or solutions. Improper sterilization Use of membrane filtration.

Airborne particles and Aerosols Equipment and Activities Laboratory Personnel

Source: www.smart-publications.com

Growing and crawling In to cultures. Top and Bottom

side walk of Dish

•Fohn Ryan, “Understanding and Managing Cell Culture Contamination,” Corning Incorporated, Corning, NY, Report No. CLS-AN-020 Rev. 2, March 8, 2008

Cross contamination from other cell cultures. Accidents Actual case demonstration

Media Major source. How they enter? Prevention

Water Leaching of metal ions, organic and

inorganic compounds. Deposition of water on instrument Prevention

SERA Sources Uncontrollable variation of

sera components Prevention

Storage vessels Material of construction Leaching Prevention: 316L stainless

steel http://www.pharmaceutical-machines.com/gifs/storage-vessel.jpg

Fluorescent Lights Exposure of media HEPES (N-[2-hydroxylethyl] piperazine-N’-

[2-ethanesulfonic acid]) – an organic buffer Production of hydrogen peroxide and free

radicals Incubators

Chemical and biological contamination source

Toxic and volatile impurities

Good Aseptic Techniques

Good Aseptic Techniques

Strategic Use of Antibiotics

Strategic Use of Antibiotics

Strategic Use of Cell Repository

Strategic Use of Cell Repository

Good Housekeeping by Everyone

Good Housekeeping by Everyone

Understanding Nature of Contamination

Understanding Nature of Contamination

Contamination Controlling Program

Contamination Controlling Program

Building Blocks for successfully managing cell culture contamination [7]

Use of good aseptic technique

Use good aseptic techniques

Reduce accidents

Keep the laboratory clean

Routinely monitor for contamination

Use frozen cell repository strategically

Use antibiotics sparingly if at all

Steps for Reducing Contamination Problems Source: Reference [7]

Vented cap flasks greatly reduce the opportunities for contamination in culture systems requiring gas exchangeSource: Reference [7]

Reduce opportunities for accidents Proper labeling Written protocol Identification and causes of contaminants

Clean up the work area and surroundings Periodical cleaning and lower level of

particulates Removal of waste materials Proper maintenance of equipment Paste control program

Sterility testing Routine monitoring of supplies Sterilization of equipments Microscopic observation of media Karyotyping, DNA fingerprinting,

Electrophoresis and immunological techniques

Detection of chemical contaminants

Strategic Use of a Frozen Cell Repository A cryogenic cell

repository Strategic use of

antibiotics Use of antibiotics Drawback of long term

and continuous use of antibiotic

Source: biorep.sevenlab.net

Implementation of single use systems Pre-sterilized assembly Reduced risk of contamination

Element Technology use Addition information Company

Disposable sensor oxygen, pH, and carbon dioxide sensors (fluromatrix technology)

LEDs & photo-detectors.

Inexpensive, useful for FDA imitative technology

Sartorius Biotechnology

Mixing Single Use Mixer Levitate and drive a disposable impeller inside a single-use bag. Scalability and reduced contamination, operating costs, and downtime associated with cleaning and validation of conventional mixing systems.

LevTech, Hyclone

Bag Flexel 3-D bag system Reduce contamination Applikon, SAFC Biosciences

Sterile sampling Integrated one-way valve that prevents any of the withdrawn cell culture media sample returning to the main bioreactor.

Reduce contamination Cellexus Sample

Disposable with Latest technologies Source: Reference [16]

New Disposable Filling System Source: Reference [11]

Rapid Molecular Method Testing standard during production Emerging as the gold standard for

contaminant and impurity analysis Based on polymer chain reactions If positive result, identify the exact

amount of mycoplasma.

Other latest tools for prevention of batch failure PAT(Process Analytical Technology)

Real-time, on-location measurement of quality control parameters.

Examples: Personal Scanning Electron Microscope (PSEM), technology by Millipore, rapid automated microbiological testing technologies

QbD (Quality by Design) “Quality cannot be tested into products; it has

to be built in by design” Approach Used to create Robust Manufacturing Process.

Other latest tools for prevention of batch failure Risk management and the CAPA

Identification of risks Classifying different types of impact Next step corrective and preventive

action FMEA (Failure Modes Effect Analysis)

Identification of effect on failure Preventive action

Production of EPO erythropoietin Serious failures in manufacturing

like QC failures for the batch Concern about the safety

Problem like Glycosylation and its control Change in manufacturing conditions

and parameters

True batch failure: final specification

False batch failure: wrong identification of aberrant glycosylation

Safety compromised: Aberrant glycosylation

Non standard efficacy: abnormal glycosylation

Solution Understand the structure and glycosylation

in life cycle Molecular specification

Structural activity relationship and other drug designing documents

Measurement specification Measurement of glycosylation parameters

Review and Modify at regular interval Parameters

Appropriate glyco analysis method Monosaccharide profile Oligosaccharide profile Glycosylation site profile Glycoform profile

Different causes of failure Large organization Small and medium organization

Eric S. Langer, “Biotech Facilities Average a Batch Failure Every 40.6 Weeks,” Bioprocess Internaional, September 2008.

By Dr Daryl Fernandes, Founder and CEO of Ludger Ltd., “Reducing Risk in Biopharmaceutical Production by Controlling Glycosylation,” European Biopharmaceutical Review, Winter 2004, pp 92 - 97.

Simon P. Langdon, “Cancer Cell Culture: Methods and Protocols,” Chap. 7, Page no. 310, Humana Press.

Eric S. Langer, “Batch Failure Rates in Biomanufacturing,” Genetic Engineering and Biotechnology News, Vol. 28, No. 14, Aug 1 2008.

Carolyn Kay Lincoln, “Cell Culture Contamination, Is It Just Mycoplasma?,” Bionique Testing Laboratories, Inc., USA, January 24, 2006.

David B. Prescott, “Methods in Cell Biology,” Chap. 6, Page No. 144-201, Vol. VI, Academic Press Inc.

Fohn Ryan, “Understanding and Managing Cell Culture Contamination,” Corning Incorporated, Corning, NY, Report No. CLS-AN-020 Rev. 2, March 8, 2008

Scott M. Wheelwright, Ph.D., “Cost drivers for the biopharmaceutical,” Strategic Manufacturing Worldwide, available on:biog.berkeley.edu/cbo/uploads/ADMIN/UCB%2017Oct07%20SMW%20v2.ppt

Patricia Seymour, “Successfully Managing Biopharmaceutical Manufacturing Outsourcing,” Pre-Conference Workshop, IQPC 4th Annual Contract Manufacturing Forum, BioProcess Technology Consultants, Inc. Acton, MA, May 22, 2006.

Ramon Rivera Gonzalez, “Quality and Compliance Challenges for Biopharmaceutical Products,” USA: Amgen Manufacturing, Limited, available on: www1.uprh.edu/micro/proyectos/BIOTECH2007.ppt

John Boehm, “A Next Step in Implementing Disposables: Transfer Lines,” BioPharm International Supplements, Nov 2, 2007 http://biopharminternational.findpharma.com/biopharm/article/articleDetail.jsp?id=473326&sk=&date=&pageID=3

Marie Vicéns, PhD, “Contamination Control | Make Contamination Detection Automatic,” Pharmaceutical formulation and Quality, by John Wiley & Sons. Inc, December/January 2009.

Kazutomo Yokoya Kdm Communications Bedford, UK, “Early Detection in Drug Production,” PharmaAsia, January 01, 2009.

Don Straus, “Rapid micro tests can improve quality and efficiency,” Carpe Diem-A Wiley Co., 111 River Street, Hoboken, NJ 07030, April 25, 2009.

Gail Dutton, “In-Line Analytics Improve Manufacturing,” Genetic Engineering & Biotechnology News, Vol. 28, No. 15, Sep 1, 2008.

“Disposable Bioreactors Gaining Favor,” Genetic Engineering & Biotechnology News, Vol. 26, No. 12, Jun 15 2006.

Peter K. Kang, PhD, “Trends in Contamination Control,” Pharmaceutical Processing, 2009, available on:http://www.pharmpro.com/ShowPR.aspx?PUBCODE=021&ACCT=0000100&ISSUE=0707&RELTYPE=PR&ORIGRELTYPE=ATO&PRODCODE=0000&PRODLETT=M&CommonCount=0

Yih-Horng Shiao, Ph.D, “Identification and Elimination of Contaminations in Cell Culture and Polymerase Chain Reaction Laboratories,” Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Maryland, USA, June 25, 2005.